1. Introduction to single-cell RNA-seq
2. Quality control and normalization
3. Survey of downstream analysis methodology

• Count distributions with overdispersion
• Variance stabilizing transformations
• Normalization for sequencing depth
• Removal of batch effects

• Heterogeneity/increased resolution
• Sparsity (few genes detected in each cell)
• New technical artifacts
  • Different protocols
  • Smaller starting material
  • ++ Multiplexing

Bulk RNA-seq measures the average expression across a pool of cells

• Detection rate = proportion of genes with nonzero measurement for that cell
• Often the vast majority of measurements are zero
• Commonly referred to as “dropout” (1-detection rate)

Hicks et al. 2018 (https://doi.org/10.1093/biostatistics/kxx053)

• platforms isolate cells, extract mRNA, and prepare libraries for sequencing
• protocols refer to the specific chemistries used to prepare sequencing libraries
• platforms may be protocol-specific (proprietary) or allow multiple protocols (open-source)

• Full-length transcript vs 3’ end only
• Plate-based vs droplet-based
• Read counts vs UMIs
• Multiplexed with barcodes vs not

Isoform analysis only possible with full-length

• lower throughput
• higher capture rate (% of input cells measured) Adamowicz, Maratou & Aitman 2017 (https://doi.org/10.1007/978-1-4939-7481-8)

• higher throughput
• lower capture rate (% of input cells measured) Macosko et al. 2015 (https://doi.org/10.1016/j.cell.2015.05.002)

• PCR introduces nonlinear amplification bias
• UMIs are a way to tag each unique molecule in the sequencing library (before PCR)
• Number of possible UMIs = \(4^L\), where \(L\) is the length of the UMI
• Low \(L\) or sequencing errors can cause barcode collisions
• Afterward, count up only the number of distinct UMIs (collapse reads)

• Need to pool together many cells per lane for high-throughput protocols
• Barcodes can be used to uniquely tag each cell
• Afterward, de-multiplex by separating read/UMI counts per barcode
• Most common in droplet protocols

image source: http://data-science-sequencing.github.io/Win2018/lectures/lecture16/

UMIs + Barcodes can be added with specially designed plastic or hydrogel beads

Macosko et al. 2015 (https://doi.org/10.1016/j.cell.2015.05.002)

• not feasible with droplet protocols

Ziegenhain et al. 2017 (https://doi.org/10.1016/j.molcel.2017.01.023)

da Veiga Beltrame et al. 2019 (https://doi.org/10.6084/m9.figshare.7704659.v1)